This invention relates to mixing fluids, and more particularly relates to an apparatus and method for mixing a small quantity of fluid that is present as a film on a solid substrate.
Methods for mixing relatively large volumes of fluids usually utilize conventional mixing devices that mix the fluids by shaking the container, by a rapid mechanical up and down motion, or by the use of a rocking motion that tilts the container filled with the fluids in a back and forth motion. The conventional mixing methods normally cannot be utilized for methods involving thin films of fluid because the capillary strength of the containment system often significantly exceeds the forces generated by shaking or rocking, thereby preventing or minimizing fluid motion in the film. The problem is illustrated by a surface chemistry reaction where the surface is large and the available fluid sample is very small. The fluid when spread across the surface results in a thin film of fluid that may have a thickness of a few microns to a few millimeters. In such situations, the fluid may not adequately contact the entire reactive surface or the reactive compounds in the fluid may be very dilute thereby resulting in a reaction that is limited by the rate of diffusion through the fluid. Thus, inadequate mixing can adversely affect the sensitivity or specificity of the reaction, the rate of reaction, the extent of reaction, the homogeneity, or the percent yield.
Inadequate mixing is a particular problem in chemical and biological assays where very small samples of chemical, biochemical, or biological fluids are typically reacted. For example, the ability to clone and synthesize nucleotide sequences has led to the development of a number of techniques for disease diagnosis and genetic analysis. Genetic analysis, including correlation of genotypes and phenotypes, contributes to the information necessary to reveal the changes in genes which confer disease. New methods of diagnosis of diseases, such as AIDS, cancer, sickle cell anemia, cystic fibrosis, diabetes, muscular dystrophy, and the like, rely on the detection of mutations present in certain nucleotide sequences. Many of these techniques generally involve hybridization between a target nucleotide sequence and a complementary probe, offering a convenient and reliable means for the isolation, identification, and analysis of nucleotides.
One typical method involves hybridization with either target or probe nucleotide sequences immobilized on a solid support. The targets or probes are usually immobilized on a solid support having a surface area of typically less than a few square centimeters. The solid support is typically a glass or fused silica slide which has been treated to facilitate attachment of either the targets or probes. The mobile phase containing reactants that react with the attached targets or probes is placed on the support, covered with another slide, and placed in an environmentally controlled chamber such as an incubator. Normally, the reactants in the mobile phase diffuse through the liquid to the interface where the complementary probes or targets are immobilized, and a reaction, such as hybridization reaction, then occurs. Preferably, the mobile phase reactants are labeled with a detectable tag, such as a fluorescent tag, so that the hybrid could be identified. The hybridization reaction typically takes place over a time period that can be many hours.
Problems are often encountered in conducting chemical or biological assays, including use of arrays, with poor hybridization kinetics and efficiency or reaction specificity and sensitivity, since diffusion is the only means of moving the reactants in the mobile phase to the interface or surface containing immobilized reactants. Alternatively, the fluid sample must be removed from the reaction chamber, mixed in separate chambers external to the reaction chamber, and then reintroduced into the reaction chamber. Valuable fluid sample is wasted or lost in the separate external chambers required in such mixing process.
A method and apparatus for mixing a thin film of fluid, particularly a chemical, biochemical, or biological fluid undergoing a reaction, is described in a copending application U.S. Ser. No. 08/889763. The application describes a thin film of fluid between two opposing surfaces that is mixed by moving one surface relative to the other.
The present invention describes an apparatus and method for mixing of a film of fluid via nucleation of bubbles within the film. The use of bubbles for mixing large volumes of liquids is well known. For example, U.S. Pat. No. 5,443,985 to Lu et al. and U.S. Pat. No. 5,605,653 to DeVos describe the mixing and aeration of large volumes of liquid, such as a culture medium in a cell culture bioreactor by introducing extraneous gas at the bottom of the reactor thereby creating bubbles that travel upwards, thus mixing the liquid medium. In another context, U.S. Pat. No. 5,275,787 to Yaguchi et al. describes the use of thermal energy to generate a bubble that is then used to discharge a sample liquid containing individual particles. The generation of the bubble and its use as an optical switching element for devices that have uses in telecommunication systems and data communication systems is described in U.S. Pat. No. 5,699,462 to Fouquet et al. and U.S. Pat. No. 4,988,157 to Jackel et al.
The invention, in one embodiment, is an apparatus for mixing a film of fluid, particularly a chemical, biochemical, or biological fluid which typically comprises a reaction mixture, the apparatus comprising a first substrate having an inner surface and a substantially parallel second substrate having an inner surface that defines a closed chamber therebetween. The closed chamber is adapted to retain a quantity of fluid so that the fluid is in contact with both inner surfaces. In addition, the apparatus comprises a means for nucleating bubbles in the fluid comprising discrete heat sources for creating individual bubbles at selected locations within the apparatus such that as each bubble is nucleated and dispelled, the fluid is displaced resulting in mixing, and a means for moving the bubbles in the fluid. The inner surface of one or both of the substrates is functionalized with reactive moieties that can react with the components contained in the fluid. The bubbles are nucleated by using discrete heat sources, such as resistors arranged in a predetermined pattern adjacent to the inner surface of the substrate.
In another embodiment, only the first substrate has the means for nucleating bubbles comprising discrete heat sources adjacent to the inner surface and is functionalized with reactive moieties.
The invention also provides a method for mixing a film of fluid comprising providing a first substrate and a substantially parallel second substrate having inner surfaces that define a closed chamber therebetween. The chamber is adapted to retain a quantity of fluid so that the fluid is in contact with both inner surfaces. The method further comprises introducing a fluid containing a plurality of components into the closed chamber so as to provide a film of fluid therein, nucleating a bubble within the fluid film, whereby, as each bubble that is nucleated is dispelled, the fluid is displaced resulting in mixing, and a means for moving a bubble in the film of fluid. The bubbles may be moved in the film of fluid by creating a temperature gradient along which the bubbles move.